Erythritol is a high quality and very low-calorie sweetness. Erythritol is also noncariotic so that it is widely used as a sweetener for food and beverage.
Several microorganisims such as Trichosporonoides and Moniliella are widely used for industrial production of erythritol from glucose. Microorganisms such as those belonging to the genera Trichosporonoides, Moniliella, etc., are caused to act on a substrate such as glucose (cf. Japanese Patent Publication No. Hei 6-30591, Japanese Patent Publication No. Hei 6-30592, Japanese Patent Publication No. Hei 4-11189, Japanese Patent Publication No. Hei 4-635, Japanese Patent Kokai No. Hei 10-96, and Japanese Patent Kokai No. Hei 9-154589).
It has been reported that erythrose reductase type I, II and III are involved in producing eythritol at Trichosporonoides megachiliensis Strain SN-G42 (FERM BP-1430) (K. Tokuoka, et al., J. Gen. Appl. Microbiol., 38, 145-155 (1992)).
The metabolic pathway from glucose to erythritol in Trichosporonoides megachiliensis Strain SN-G42 is illustrated in FIG. 1.
As illustrated in FIG. 1, glucose enters the Pentose Phosphate Shunt to produce erythrose-4-phosphate (Erythrose-4-P) after this sugar phosphate is metabolited to glucose-6-phosphate (Glc-6-P) or glyceraldehyde-3-phosphate by glycolysis.
The erythrose-4-phosphate is dephosphorylated to produce D-erythrose and D-erythrose, which gets reduction by NADPH or NADH to produce meso-eythritol.
Of such a series of reactions, the erythrose reductase type I, II and III all catalyze the latter reductive reaction (i.e., the reaction in which erythrose gets reduction by NADPH or NADH to form meso-eythritol).
The reports on erythrose reductases only described their enzymological properties. The genetical analysis of these enzymes remains to be elucidated.
An object of the present invention is to provide an efficient method for the production of erythritol.
Another object of the present invention is to clarify the primary structures of enzyme having an erythrose reductase activity and to characterize a complementary DNA encoding the protein in order to establish an erythritol producing microorganism and to provide a method for utilizing them.
If it is successful in obtaining the DNA that encodes the protein having an erythrose reductase activity, a large amount of proteins can be produced by expressing the DNA in a cell such as Esherichia coli, yeast cell, etc., or the like means. This invention not only leads to mass production of erythritol but also is applied to development of mutant enzymes having higher erythritol productivity, cloning of DNA encoding related enzymes and the like by using genetic engineering techniques.